MOUSE ANTI FELINE CORONAVIRUS Antibody (0.25mg)

Catalog#: OASA00695

• Size: 0.25mg

• Description of Target: MOUSE ANTI FELINE CORONAVIRUS
• Alias Symbols: N/A
• Host: Mouse
• Specificity: CORONAVIRUS
• Application: IHC-AFF, ELISA, FC, IF, IHC-FFPE, WB
• Clone: FIPV3-70
• Immunogen: Coronavirus cocktail
• Product Format: Purified IgG - liquid
• Isotype: IgG2a
• Applications Info: (1) Special conditions apply - see (*)
• Predicted Homology Based on Immunogen Sequence: Viral
  Preparation: Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
  
  Preservative Stabilisers: 0.05% - Sodium Azide
  Approx Protein Conc: IgG concentration 1.0 mg/ml
  
  Buffer Solutions: Phosphate buffered saline pH 7.2
• Reconstitution and Storage: Store at +4^oC or at -20^oC if preferred.
  
  This product should be stored undiluted.
  
  Storage in frost-free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
• Key Reference: 1. Kipar, A. et al. (1998) Fatal enteritis associated with coronavirus infection in cats. J. Comp. Path. 119: 1 - 14.
  2. Kipar, A. et al. (2000) Expression of viral proteins in feline leukemia virus-associated enteritis. Vet. Pathol. 37: 129 - 136.

Product Protocol: MOUSE ANTI FELINE CORONAVIRUS antibody used to evaluate detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis (OASA00695)

Specificity: CORONAVIRUS
Product Page: MOUSE ANTI FELINE CORONAVIRUS antibody (OASA00695)
Antibody Pair Available: OASA00695 Capture OASA00696 Detection
Experiment Type: Immunohistochemical techniques, Reducing Western blots

Protocol:
1. Immunohistochemical techniques:

FIPV3-70 and CCV2-2 mAbs were obtained from Custom Monoclonals International (West Sacramento, CA). Both mAbs were made by immunizing Balb/c mice with canine coronavirus (CCoV, strain CCV51). The epitopes recognized by these antibodies are unknown. The original concentration of these mAbs was 4.5 mg/ml and 4.1 mg/ml, respectively. At least 2 tissue blocks exhibiting typical histopathologic changes were selected for IHC investigations from each cat. Adjacent sections were treated with either FIPV3-70 or CCV2-2 mAbs; in addition, sections from both positive and negative (FIPV3-70 or CCV2-2 mAbs treated) cases were also tested with MAC 387 antihuman myeloid/histiocyte antigen mAb (Serotec, Oxford, UK).

For optimal FIPV3-70 mAb staining, L. Poncelet et al. found it best to preheat sections (close to boiling point) in 0.01 M citrate buffer for 20 minutes in a microwave before the primary antibody. CCV2-2 mAb did not require any pretreatment for epitope retrieval.

MAC 387 antibody reaction required 15 minutes of preincubation at 37°C with 0.1% trypsine and 0.1% CaCl2 solution (as suggested by the supplier).

Dewaxed sections were rinsed; nonspecific binding was blocked by incubating the slides with 1/20 normal sheep serum (NSS) for 15 minutes. Incubation with the primary mAb (FIPV3-70 diluted to 1/400, CCV2-2 at 1/600, and MAC 387 at 1/1000 all in 1/100 NSS) occurred for 12 to 36 hours at 4°C. Sections were rinsed, incubated with 1/20 NSS, and then with goat antimouse immunoglobulin serum at 1/80 in 1/100 NSS as secondary antibody for 15 minutes. Slides were rinsed then incubated for 15 minutes with peroxidase-antiperoxidase complex 1/300 in 0.01 M PBS for 15 minutes. After rinsing, the presence of antigen was revealed with DAB-H2O2 solution (liquid DAB substrate pack from Biogenex, San Ramon, CA), which resulted in a brown deposit. The sections were lightly counterstained with hematoxylin, dehydrated, and mounted.

Sections from up to 10 blocks, negative for FIPV3-70 mAb, were further processed. Thus, sections were first screened immunohistochemically using FIPV3-70 as the primary antibody. Then, CCV2-2 mAb was applied (as the other primary antibody) on the same section. This second reaction was revealed using DAB in Tris/Nickel buffer with 0.1% H2O2 as chromogen, which resulted in blue staining. These successive immunostainings were also conducted with the same mAbs used in reverse order, that is, CCV2-2 first followed by FIPV3-70.

Double immunofluorescent staining was also carried out on tissue blocks positive for both mAbs on adjacent sections, using biotinylated horse antimouse secondary antibody (1/100 in 1/100 NSS), after blocking the endogenous biotin (Avidin/biotin blocking kit, Vector, Burlingame, CA). Color development was conducted with Alexa 594–coupled streptavidine after incubation with FIPV3-70 mAb (red fluorescence) and with Alexa 488 coupled–streptavidine after incubation with CCV2-2 mAb (green fluorescence). Both fluorochromes were obtained from Molecular Probe (Carlsbad, CA) and diluted at 1/100 in 0.01 M PBS. Slides were mounted with an aqueous medium (Vectashield, Vector) and analyzed with a fluorescent microscope.

In all immunostaining runs, slides in which the primary antibody step was skipped or replaced by an irrelevant mAb served as negative controls. Positive controls were also systematically included: sections from the serosal lining of the control cat with the abdominal exudative FIP (this tissue stained positive for both FIPV3-70 and CCV2-2 mAbs) for FCoVstaining and sections from a feline mesenteric lymph node for MAC 387 staining.

2. Reducing Western blots:

The Western blot technique was used to evaluate the specificity of the 2 mAbs on 2 sources of viral antigens: 1 from a laboratory strain of the CCoV (isolate CCV51 adapted to grow in Crandell feline kidney [CrFK] cells, a gift from L. E. Carmichael at the Barker institute for Animal Health, Cornell University, to C. K. G.) and the other from a field virus from a diseased cat.

The CCV51 coronavirus isolate was grown in confluent monolayers of CrFK cells, and the virus plus cell debris was harvested at the stage of syncytial cell formation. The culture mixture was snap frozen and thawed and then spun at 1,500 × g to remove cellular debris; clarified supernatant was then ultracentrifuge pelleted (110,000 × g for 90 minutes using a swinging bucket rotor). The virus pellets so obtained were pooled, washed once using PBS pH 7.4, and then resuspended in PBS (positive control). For noninfected CrFK membranes, uninfected cell cultures were treated identically (negative control).

For the feline virus, frozen granulomatous serosal lining from the control cat with the typical form of abdominal exudative FIP was processed. This same tissue tested positive (FIP+) with both mAbs, namely FIPV3-70 and CCV2-2, in IHC preparations.

Serosal lining from a healthy control cat was also collected, which tested negative (FIP-) for the same mAbs.

The frozen samples from the healthy and diseased control cats were mechanically homogenized in 5 volumes of a Tris-HCL buffer (10 mM, ph 7.4) containing 5 μl/ml aprotinin 10% (Sigma, Providence, RI) and 5 μl/ml phenylmethylsulfonyl fluoride (1.74 mg/ml isopropanol). Total protein was measured using a colorimetric procedure (BCA Protein Assay Kit, Pierce, Rockford, IL) and was found to be 2.65 mg/ml and 13.7 mg/ml for the positive and negative control preparations, respectively, and 101 mg/ml and 97 mg/ml for FIP+ and FIP- samples, respectively.

Virus and negative control preparations were diluted in 4 volumes of Tris-HCL buffer (0.5 M, pH 6.8) containing 0.1 g/ml sodium dodecyl sulfate (SDS; Sigma), 0.1 mg/ml beta mercaptoethanol 20%, 0.5 g sucrose, and 0.1 ml bromophenol blue and were boiled for 5 minutes. The total amount of proteins loaded on a gel-loading surface was 150 μg for FIP+, 162 μg for FIP-, 40 μg for negative control, and 5.76 μg for positive control.

The nonspecific lane was loaded with FIP+ (150 μg). Samples were subjected to electrophoresis (75 minutes, 150 V) in a Tris-glycine discontinuous SDS-polyacrylamide gel (4 % stacking gel, 10% separating gel). After semidry protein transfer (60 minutes, 100 mA), the nitrocellulose membranes were saturated for 2 hours in Tris-HCl buffer (10 mM, pH 7.4) containing 10% powdered fat-free milk and 0.2% Tween 20 (Sigma). The blots were incubated (in 15 ml/blot) overnight with either FIPV3-70 or CCV2-2 mAb diluted (15 μg/ml) in 2% bovine serum albumin buffer (BSA; Sigma). The nonspecific control blot lane was incubated in the 2% BSA buffer. The blots were washed 4 times, 15 minutes each, with 0.2% Tween 20 buffer and incubated for 2 hours (15 ml/blot) with 1/2000 antimouse IgG alkaline phosphatase conjugate (Sigma) in 2% BSA buffer. Staining was performed in 100 mM Tris (pH 9.5) containing 100 mM NaCl, 5 mM MgCl2, bromochloroindolyl phosphate (3.5 μl/ml), and nitro blue tetrazolium substrates (4.5 μl/ml) (Sigma).

Molecular weights of proteins recognized by the mAbs were determined by comparison with either prestained low- and high-range molecular weights standards (Amersham, Life Sciences, UK) or the SDS-6B molecular weight range (Sigma).

Summary:
1. Reducing Western blots revealed that both anti-CCoV mAbs (FIPV3-70 and CCV2-2) reacted with a protein of approximately 50 kD, when the virus was ultracentrifuge sedimented from CCoV-infected cell culture supernatant, and also with a similar sized band when the virus was derived from infected cat serosal tissues. This molecular weight is compatible with the FCoV nucleocapsid protein (N). 2. FIPV3-70–positive cells (also with monocyte/macrophage morphology) were observed in only 7 of the 15 cats. In addition, in 5 of the 7 FIPV3-70–positive cats, not all cerebral structures were positive for FIPV3-70.

References:
1: Poncelet L, Coppens A, Peeters D, Bianchi E, Grant CK, Kadhim H. Detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis. Vet Pathol. 2008 Mar;45(2):140-53. PubMed PMID: 18424826.